The repressor protein of bacteriophage Mu establishes and maintains lysogeny by shutting down transposition functions needed for phage DNA replication. It interacts with several repeated DNA sequences within the early operator, preventing transcripti ...

The repressor protein of bacteriophage Mu establishes and maintains lysogeny by shutting down transposition functions needed for phage DNA replication. It interacts with several repeated DNA sequences within the early operator, preventing transcription from two divergent promoters. It also directly represses transposition by competing with the MuA transposase for an internal activation sequence (IAS) that is coincident with the operator and required for efficient transposition. The transposase and repressor proteins compete for the operator/IAS region using homologous DNA-binding domains located at their amino termini. Here we present the solution structure of the amino-terminal DNA-binding domain from the repressor protein determined by heteronuclear multidimensional nuclear magnetic resonance spectroscopy. The structure of the repressor DNA-binding domain provides insights into the molecular basis of several temperature sensitive mutations and, in combination with complementary experiments using flourescence anisotropy, surface plasmon resonance, and circular dichroism, defines the structural and biochemical differences between the transposase and repressor DNA-binding modules. We find that the repressor and enhancer domains possess similar three-dimensional structures, thermostabilities, and intrinsic affinities for DNA. This latter result suggests that the higher affinity of the full-length repressor relative to that of the MuA transposase protein originates from cooperative interactions between repressor protomers and not from intrinsic differences in their DNA-binding domains. In addition, we present the results of nucleotide and amino acid mutagenesis which delimits the minimal repressor DNA-binding module and coarsely defines the nucleotide dependence of repressor binding.

Organizational Affiliation:&nbsp

Department of Chemistry and Biochemistry, UCLA-DOE Laboratory of Structural Biology and Genetics, University of California-Los Angeles 90095-1570, USA.